Anti-viral compounds

ABSTRACT

The present invention relates generally to compounds useful in the amelioration of symptoms associated with viral infection. More particularly, the present invention relates to the use of compounds which exhibit a physiological effect on membranous and/or transmembranous structures on or in a cell and which directly or indirectly reduce or inhibit or otherwise prevent viral infection, processing and/or release from the cell. Even more particularly, the present invention contemplates the use or one or more compounds which modulate at least one host cell ion channel in the prophylaxis, treatment and/or symptomatic relief of viral infection in vertebrate animals and in particular in human subjects. The compounds may be provided alone or in combination with other compounds such as those which block or inhibit or at least impair ion channeling. A preferred embodiment of the present invention is the use of the aforementioned anti-viral compounds in the therapeutic management of vertebrate animals including humans, to prevent, reduce or treat infection by certain species of the Picornaviridae family of viral pathogens such as but not limited to  Rhinovirus  or  Enterovirus  species.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compounds useful in theamelioration of symptoms associated with viral infection. Moreparticularly, the present invention relates to the use of compoundswhich exhibit a physiological effect on membranous and/ortransmembranous structures on or in a cell and which directly orindirectly reduce or inhibit or otherwise prevent viral infection,processing and/or release from the cell. Even more particularly, thepresent invention contemplates the use of one or more compounds whichmodulate at least one host cell ion channel in the prophylaxis,treatment and/or symptomatic relief of viral infection in vertebrateanimals and in particular in human subjects. The compounds may beprovided alone or in combination with other compounds such as thosewhich block or inhibit or at least impair ion channeling. A preferredembodiment of the present invention is the use of the aforementionedanti-viral compounds in the therapeutic management of vertebrate animalsincluding humans, to prevent, reduce or treat infection by certainspecies of the Picornaviridae family of viral pathogens such as but notlimited to Rhinovirus or Enterovirus species.

2. Description of Thee Prior Art

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the corninon general knowledge in any country.

The development of medicines to treat viral pathogens has been a goal ofhealth researchers worldwide for many years, but despite close attentionto the task there remains a need for effective anti-viral drugs for theefficacious therapeutic management of a vast number of viral-mediatedillnesses.

There are numerous problems which are encountered in the search fordrugs that will effectively inhibit the spread of viruses in a hostorganism. Briefly, these include the ability of many viruses to avoidactivating the immune system's detection mechanism, the capacity ofviruses to replicate and spread prior to being detected by the immunesystem and the high mutation rate of viral proteins.

The Picomaviridae family is a ubiquitous group of viral pathogens thatcauses the most common, and often serious, viral-mediated illnesses inhumans. The range of diseases caused by this group includes ascepticmeningitis, poliomyelitis, certain types of myocarditis as well asrhinovirus infection. Rhinoviruses cause over 80% of all cases of acutenasopharyngitis (the common cold) [Monto et al., Clin. Ther. 10:1615-27, 2001], and hence are the cause of the one respiratory infectionwhich creates the most restriction of activity and necessitates thegreatest number of physician consultations per year in Western nations.

The Picornaviridae family is also of veterinary significance: theAphthovirus genus is responsible for the recent, economicallydevastating foot-and-mouth disease in livestock animals.

Hence, due to its ability to infect humans and livestock animals alike,this group of pathogens represents a significant economic liabilityworldwide.

New drugs such as Pleconaril have been developed with the specificpurpose of treating rhinovirus- and other types of picornavirusinfections, but a significant drawback of these drugs is that there isthe potential for the formation of Pleconaril-resistant viral strains(Turner, Antiviral Res. 49(1): 1-14, 2001). This is a consequence of thehigh capacity of viruses to mutate and overcome the effects ofanti-viral compounds that have been designed to inhibit one or moresteps in the process of the virus's entry into a host cell and itssubsequent replication in the cell.

Research has found that infection by certain members of thePicomaviridae family cause alterations in the intracellular ion levelsof infected cells. In particular, poliovirus and Coxsackievirus causeincreases in the cytoplasmic concentration of calcium (Irurzum et al.,J. Virol. 69(8): 5142-6, 1995; van Kuppeveld et al., EMBO J. 16(12):3519-32, 1997) and encephalomyelitis virus infection as well aspoliovirus infection have both been found to disrupt cellular sodium andpotassium-ion homeostasis (Egberts et al., J. Virol. 22(3): 591-7, 1977;Nair, J. Virol. 37(1): 268-73,1981; Nair et al., J. Virol. 31(1): 184-9,1979). The net result of these alterations in ionic transport appears tobe an influx of sodium and/or calcium ions into the cell cytoplasm.

In accordance with the present invention, compounds are identified whichalter the permeability of ion channels in the host cell and,surprisingly, found that these compounds were effective in controllingthe replication and/or spread of viruses and in particular of members ofthe Picornaviridae family.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The present invention provides a method for the treatment or prophylaxisof viral infection in vertebrate animals such as but not limited tohumans, livestock animals, avian species, companion animals, andlaboratory test animals. The compounds are generally defined by theirability to modulate the activity of a membranous or transmembranousstructure which permits passage of ions into or out of a vertebrateanimal cell.

In a preferred embodiment, these membranous or transmembranousstructures are referred to as ion channels and the preferred compoundsare generally regarded as “ion channel blockers” or “ion channelmodulators”. Such compounds are proposed inter alia to alter thefunctional properties of an ion channel such as creating an open-channelstate (complete activation), partial activation, inhibition or totalblockage of the ion channel.

The compounds contemplated in the present invention are represented inFormulae I through IV as herein defined. The preferred compounds areVerapamil (Formula V), Econazole (Formula VI), Benzamil (Formula VII)and 5-(N-ethyl-N-isopropyl)amiloride [EIPA] (Formula VIII) and Amiloride(Formula IX), the parent compound of EIPA. Reference to all suchcompounds include pharmaceutical salts thereof as well as derivativesthereof.

The compounds may be administered singularly or in combination with eachother or with compounds having antiviral properties, ionchannel-blocking properties or compounds which otherwise facilitateamelioration of the symptoms of viral infection.

The compounds may, therefore, be in the form of a pharmaceuticalcomposition comprising the compound and one or more pharmaceuticallyacceptable carriers and/or diluents.

The viruses are generally members of the Picomaviridae family such asbut not limited to species of Rhinovirus or Enterovirus.

In a preferred embodiment, the compounds are administered to a subjectfor a time and under conditions sufficient to ameliorate the symptoms ofviral infection or to prevent or reduce viral infection.

The present invention further provides for the use of a compound ofgeneral Formulae I-IV or more specifically Verapamil, Econazole,Benzamil and/or EIPA as well as its parent compound Amiloride in themanufacture of a medicament for the treatment or prophylaxis of viralinfection in a vertebrate animal such as a human. EIPA and Verapamilwere determined to be particularly effective against Rhinovirus.Amiloride and Benzamil are particularly effective against Enterovirus.

Reference to the “compounds” of the present invention means the generalcompounds of Formulae I-IV and the specific compounds in Formulae V-IX.The term “compounds” also encompasses “chemical agents” as well as“therapeutic agents” and “active ingredient” and “active”.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the effect of5-(N-ethyl-N-isopropyl)amiloride [EIPA] on Rhino 2 virus production andHeLa cells. Virus production (-Δ-) and cell metabolism (-o-) weremeasured against concentration of EIPA (in μM).

FIG. 2 is a graphical representation of the effects of Verapamil onRhino 14 virus production and HeLa cells metabolism. Virus product (-Δ-)and cell metabolism (-o-) were measured against concentration of EIP (inEM).

FIG. 3 is a graphical representation of the effects of EIPA on Rhino 2virus production and HeLa cells. Virus production (-Δ-) and cellmetabolism (-o-) were measured against Verapamil (in EM).

FIG. 4 is a graphical representation of the effects of Verapamil onRhino 14 virus production and HeLa cells. Virus production (-Δ-) andcell metabolism (-o-) were measured against Verapamil (in EM).

FIG. 5 is a graphical representation of the effects of Amiloride onCoxsackievirus B3 production and HeLa cells. Virus production (-Δ-) andcell metabolism (-o-) were measured against Amiloride (in μM).

FIG. 6 is a graphical representation of the effects of Benzamil onCoxsackievirus B3 production and HeLa cells. Virus production (-Δ-) andcell metabolism (-o-) were measured against Benzamil (in μM).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the use of compoundswhich exhibit a physiological effect on membranous or transmembranousstructures on or in a cell to reduce the virulence of a viral pathogenfor the prophylaxis, treatment and/or symptomatic relief of viralinfections in vertebrate animals and in particular humans. A vertebrateanimal includes livestock animals, avian species, companion animals,laboratory test animals as well as humans. Humans are particularlypreferred.

The term “virulence” in this context includes the ability of the virusto undergo processing in a host cell to generate virus particles.

The term “processing” includes attachment and penetration of viralparticles or viral nucleic acid molecules into or onto a cell, viralnucleic acid replication, synthesis of viral-derived proteins andassembly and release of viral particles.

The term “pathogen” includes any virus, whether generally consideredpathogenic or not, which causes or at least facilitates a level ofinfection which induces symptoms of infection.

A “symptom” includes a visible symptoms such as ill health or physicalsigns of infection or infection identified by, for example,immunological testing.

Accordingly, one aspect of the present invention contemplates a methodfor ameliorating the effects of Picomaviridae infection in a vertebrateanimal said method comprising administering to said animal an effectiveamount of one or more compounds selected from the compounds of FormulaeI-IV or a parent of these compounds:

where n is 0-10 atoms and both n and X may be the same or different andeach is selected from carbon, oxygen, nitrogen, sulfur, phosphorus,silicon, boron, arsenic and selenium;

-   -   R₁ to R₂₃ may be the same or different and each is selected from        hydrogen, F, Cl, Br, I, CN, NC, NO₂, CF₃, COR₁, CO₂R₁, OR₁, SR₁,        NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂,        SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂, (C═X)R₁        or X(C═X)R₁ where X is selected from sulfur, oxygen and        nitrogen; C₁-C₂₀ alkyl (branched and/or straight chained),        C₁-C₂₀ arylalkyl, C₃-C₈ cycloalkyl, C₁-C₁₀ aldoxy, C₁-C₁₀ alkyl        carbonyl, C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle,        C₂-C₁₀ alkenyl, C₁-C₁₀ heteroarylalkyl, C₁-C₁₀ alkoxyalkyl,        C₁-C₁₀ haloalkyl, dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀        [CN, NC, OR₁, SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁,        SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃,        B(R₁)₂]alkyl; aryl is C₆-C₁₄ with any mode of substitution        containing F, Cl, Br, I, NO₂, CF₃, CN, NC, COR₁, CO₂R₁, OR₁,        SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂,        SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂]alkyl.        Heteroaryl is oxazolyl, thiazaoyl, thienyl, furyl,        1-isobenzofuranyl, 3H-pyrrolyl, 2H-pyrrolyl, N-pyrrolyl,        imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl,        pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl,        indoyl, indolyl, purinyl, phthalazinyl, 1,2,3-triazolyl,        1,2,4-triazolyl, 1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl,        1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,        1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl,        1,2,3-triazinyl, azepinyl, oxepinyl, thiepinyl, benzofuranyl,        isobenzofuranyl, thionaphthenyl, isothionaphthenyl, indoleninyl,        2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl,        isoindazolyl, indoxazinyl, benzoxazolyl, anthranilyl,        quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl,        naphthyridinyl, pyrido[3,4-b]pyridinyl, pyrido[3,2-b]pyridinyl,        pyrido[4,3-b]pyridinyl.

Generally the administration of the compounds is for a time and underconditions sufficient to reduce the amount of virus replication and/orviral release from cells exposed to said compounds. Alternatively, or inaddition, the administration is for a time and under conditions for areduction or amelioration of symptoms of infection.

The compounds of the present invention may also be administered withpharmaceutically acceptable compatible counterions such as salts formedwith acids, including but not limited to hydrochloric, sulphuric,acetic, lactic, tartaric, malic and succinic acids.

As used herein the term “alkyl” refers to linear or branched chainedsaturated, aliphatic hydrocarbon groups having a specific number ofcarbon atoms. The term “haloalkyl” refers to an alkyl group substitutedby at least one halogen. Similarly, the term “haloalkoxy” refers to analkoxy group substituted by at least one halogen. As used herein theterm “halogen” refers to fluorine, chlorine, bromine and iodine. AC₂-C₁₀ alkynyl or C₂-C₁₀ alkenyl may comprise a branched or straightchained aryl and/or heteroaryl attached groups.

As used herein the term “aryl” refers to aromatic carbocyclic ringsystems such as phenyl or naphtyl, anthracenyl, especially phenyl.Suitably, aryl is C₆-C₁₄ with mono, di- and tri-substitution containingF, Cl, Br, I, NO₂, CF₃, CN, OR₁, COR₁, CO₂R₁, NHR₁, NR₁R₂, NR₁OR₂,ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂, P(R)₃,P(═O)(R)₃, Si(R₁)₃, BR₂, wherein R₁ and R₂ are as defined for R₁-R₂₃.

As used herein, “haloalkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, substituted with 1 or more halogen(for example—CxFy where x=1 to 3 and y=1 to (2x+1)); “aldoxy” representsan alkyl group with an indicated number of carbon atoms attached throughan oxygen bridge; “cycloalkyl” is intended to include saturated ringgroups, including mono-, bi- or poly-cyclic ring systems, such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyland cyclooctyl. A cycloalkyl includes a “biycloalkyl” includes saturatedbicyclic ring groups such as [3.3.0] bicyclooctane,[4.3.0]bicyclononane, [4.4.0] bicyclodecane (decalin), [2.2.2]bibyclooctane, and so forth. “Alkenyl” is intended to includehydrocarbon chains of either a straight or branched configuration andone or more unsaturated carbon-carbon bonds which may occur in anystable point along the chain, such as ethenyl, propenyl, and the like;and “alkynyl” is intended to include hydrocarbon chains of either astraight or branched configuration and one or more triple carbon-carbonbonds which may occur in any stable point along the chain, such asethynyl, propynyl, and the like. “Alkylcarbonyl” is intended to includean alkyl group of an indicated number of carbon atoms attached through acarbonyl group to the residue of the compound at the designatedlocation. “Alkylcarbonyloxy” is intended to include an alkyl group of anindicated number of carbon atoms attached to a carbonyl group, where thecarbonyl group is attached through an oxygen atom to the residue of thecompound at the designated location.

“Halo” or “halogen” as used herein refers to fluoro, chloro, bromo andiodo; and “counterion” is used to represent a small, negatively chargedspecies such as chloride, bromide, hydroxide, acetate, sulfate and thelike.

The term “substituted” as used herein means that one or more hydrogenson the designated atom is replaced with a selection from the indicatedgroup, provided that the designated atom's normal valency is notexceeded, and that the substitution results in a stable compound.

As used herein the terms “heterocycle”, “heterocyclic”, “heterocyclicsystems” and the like refer to a saturated, unsaturated or aromaticcarbocyclic group having a single ring, multiple fused rings (forexample, bicyclic, tricyclic or other similar bridged ring systems orsubstituents), or multiple condensed rings, and having at least oneheteroatom such as nitrogen, oxygen or sulfur within at least one of therings. This term also includes “heteroaryl” which refers to aheterocycle in which at least one ring is aromatic. Any heterocyclic orheteroaryl group can be unsubstituted or optionally substituted with oneor more groups, as defined above. Further, bi- or tricyclic heteroarylmoieties may comprise at least one ring, which is either completely orpartially saturated. Suitable heteroaryl moieties include but are notlimited to oxazolyl, thiazaoyl, thienyl, furyl, 1-isobenzofuranyl,3H-pyrrolyl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl,isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl,indolizinyl, isoindolyl, indoyl, indolyl, purinyl, phthalazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl,azepinyl, oxepinyl, thiepinyl, benzofuranyl, isobenzofuranyl,thionaphthenyl, isothionaphthenyl, indoleninyl, 2-isobenzazolyl,1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, isoindazolyl, indoxazinyl,benaoxazolyl, anthranilyl, quinolinyl, isoquinolinyl, cinnolinyl,quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl andpyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl.

Heterocyclic systems include partially and fully saturated heteroarylderivatives. Heterocyclic systems maybe attached to another moiety viaany number of carbon atoms or heteroatoms of the radical and are bothsaturated and unsaturated.

It is proposed the compounds defined in Formulae I-IV or a parent of oneor more of these compounds induce a physiological effect on membranousand/or transmembranous structures. In particular, and not intending tolimit the present invention to any one theory or mode of action, thecompounds of Formulae I-IV or a parent compound thereof are consideredto block or otherwise impair the function or activity of ion channels.This effect is loosely encompassed in the term “ion channel blockers”,however, the term “blockers” is not to necessitate total blockage orprevention of ion channel activity. The term “ion channel modulators”may also be used.

Reference herein after to “ion channels” includes but is not limited tothe following classes of membrane-bound ion channels and all theirisoforms:

-   -   Sodium Channels, including: voltage-gated Na⁺ channels;        non-voltage-gated Na⁺ channels; Na⁺/H⁺ exchangers; Na⁺-glucose        transporters; Na⁺/myosinositol cotransporters; Na⁺/iodide        symporters; Na⁺-dependent multivitamin transporters;        voltage-gated Ca²⁺ channels, which act as voltage sensors and as        Ca²⁺-selective pores (this channel-type includes L-type Ca²⁺        channels which are located in skeletal muscle, brain, cardiac        muscle, neuroendocrine organs and in the retina; N-type Ca²⁺        channels which are presynaptic and involved in neurotransmitter        release; P-type Ca²⁺ channels which are involved in the release        of neurotransmitter at neuromuscular junctions; Q-type Ca²⁺        channels; R-type Ca²⁺ channels; and T-type Ca²⁺ channels);        capacitive Ca²⁺ entry channels; ligand gated Ca²⁺ entry channels        (eg. Ca²⁺ transporting ATPases); intracellular Ca²⁺ channels,        including: RYR1, RYR2, RYR3, nicotinic acid adenine dinucleotide        phosphate (NAAP) receptors, sphingolipid receptor (EDG1) and IP3        receptors, which act as intracellular Ca²⁺ release channels;        Ca²⁺ sensors; voltage-gated K⁺ channels; inward rectifier K⁺        channels; delayed rectifier K⁺ channels; Ca²⁺ sensitive K⁺        channels (high conductance, intermediate conductance and small        conductance); ATP-sensitive K⁺ channels; sodium-activated K⁺        channels; cell volume sensitive K⁺ channels; type A K⁺ channels;        receptor-coupled K⁺ channels.

In a preferred embodiment, the host cell ion channels that will beaffected by the ion channel modulators are from the Ca²⁺/Na⁺ exchangers,Na⁺ H⁺ exchangers, ligand- and voltage-gated Ca²⁺ channels andstore-operated Ca²⁺ channels.

In a preferred embodiment, the compounds encompassed in Formulae I-IVare Verapamil (Formula V), Econazole (Formula VI), Benzamil (FormulaVII) and or 5-(N-ethyl-N-isopropyl)amiloride [EIPA] (Formula VIII):

Another preferred compound is Amiloride (Formula IX) which is the parentcompound of EIPA:

Accordingly, a preferred aspect of the present invention provides amethod for ameliorating the effects of Picomaviridae infection in avertebrate animal, said method comprising administering to said animalan effective amount of one or more compounds selected from Verapamil,Benzamil, Econazol, 5-(N-ethyl-N-isopropyl)amiloride [EIPA] andAmiloride or derivatives thereof and/or pharmaceutically acceptablesalts thereof.

Reference herein to Verapamil, Benzamil, Econazol, EIPA and Amilorideinclude their derivatives. Examples of such derivatives are preferablycompounds which fall within the scope of the compounds of generalFormulae I-IV.

The vertebrate animal is as defined above and includes a human.

Accordingly, another aspect of the present invention is directed to amethod for the prophylaxis or treatment of infection by a virus of thePicomaviridae family in a vertebrate animal, said method comprisingadministering to said animal an effective amount of a compound selectedfrom Verapamil, Benzamil, Econazol, EIPA and Amiloride orpharmaceutically acceptable salts thereof and/or derivatives thereofwherein said derivative is selected from compounds within generalFormulae I-IV, as herein defined.

Verapamil and EIPA are particularly useful against Rhinovirus.

Amiloride and Benzamil are particularly effective against Enterovirus.

The effect of these compounds is preferably but not exclusively toinduce ion channel modulation and/or exhibit ion channel-modulatingproperties.

The terms “ion channel modulation” and “ion channel modulatingproperties” as contemplated herein includes an ability to alter thefunctional properties of an ion channel such as creating an open-channelstate (complete activation), partial activation, inhibition and totalblockage of the ion channel. Ion channel modulation may be induced byion channel blockers or modulators.

The compounds may be used alone or in combination with each other and/orin combination with other ion channel blockers. Other host-cellion-channel blocking agents contemplated by the present inventioninclude but are not limited to: Na⁺ channel blockers: Tetrodotoxin,Saxitoxin, conotoxins, scorpion toxins, sea anemone toxins,Batrachotoxin, Ciguatoxin, Grayanotoxin, Lidocaine, Phenyloin,Amiloride, Benzamil, EIPA; Ca²⁺ channel blockers: dihydropyridines (e.g.nifedipine), phenylalkylamines (e.g. Verapamil), benzothiazepines (e.g.Diltiazem), Calciseptine, Agotoxin, SNX-325 (Segestra spider toxin),SNX-482 (Hysteroscates gigas spider toxin), nickel ions, Mibefradil,Kurtoxin, conotoxins, Econazole, EIPA; inward rectifier K⁺ channelblockers: LY97241, Gaboon viper venom, Sr²⁺, Ba²⁺, Cs²⁺; delayedrectifier K⁺ channel blockers: 4-aminopyridine, dendrotoxins,Phencyclidine, Phalloidin, 9-Aminoacridine, Margatoxin, imperator toxin,Charybdotoxin; high-conductance Ca²⁺-sensitive K⁺ channel blockers:Iberiotoxin, (+)-Tubocurarine, Charybdotoxin, Noxiustoxin, Penitrem-A,TEA; intermediate conductance Ca²⁺-sensitive K⁺ channel blockers:Cetiedil, Trifluoroperazine, Haloperidol; small conductanceCa²⁺-sensitive K⁺ channel blockers: Aparnin, Leiurotoxin 1,(+)-Tubocurarine.

Viruses contemplated in the Picornaviridae family include but are notlimited to: Genus Virus name (synonym) followed by (acronym) Enterovirusbovine enterovirus 1 (BEV-1) bovine enterovirus 2 (BEV-2) humancoxsackievirus A 1 to 22 (CAV-1 to 22) human coxsackievirus A 24(CAV-24) human coxsackievirus B 1 to 6 (CBV-1 to 6) human echovirus 1 to7 (EV-1 to 7) human echovirus 9 (EV-9) human echovirus 11 to 27 (EV-11to 27) human echovirus 29 to 33 (EV-29 to 33) human enterovirus 68 to 71(HEV68 to 71) human poliovirus 1 (HPV-1) human poliovirus 2 (HPV-2)human poliovirus 3 (HPV-3) porcine enterovirus 1 to 11 (PEV-1 to 11)simian enterovirus 1 to 18 (SEV-1 to 18) Vilyuisk virus Rhinovirusbovine rhinovirus 1 (BRV-1) bovine rhinovirus 2 (BRV-2) bovinerhinovirus 3 (BRV-3) human rhinovirus 1A (HRV-1A) human rhinovirus 1 to100 (HRV-1 to 100) Hepatovirus hepatitis A virus (HAV) simian hepatitisA virus (SHAV) Cardiovirus encephalomyocarditis virus (EMCV) (ColumbiaSK virus); (mengovirus) (mouse Elberfield virus) Theiler’s murineencephalomyelitis virus (TMEV) (murine poliovirus) Aphthovirusfoot-and-mouth disease virus A (FMDV-A) foot-and-mouth disease virusASIA 1 (FMDV-ASIA1) foot-and-mouth disease virus C (FMDV-C)foot-and-mouth disease virus O (FMDV-O) foot-and-mouth disease virus SAT1 (FMDV-SAT1) foot-and-mouth disease virus SAT 2 (FMDV-SAT2)foot-and-mouth disease virus SAT 3 (FMDV-SAT3) Parechovirus Humanparechovirus Erbovirus Equine rhinitis B virus Kobovirus Aichi virusTeschovirus Porcine teschovirus

Accordingly, present invention is based on the use of substances withion-channel blocking properties to reduce the virulence of aPicomaviridae virus, for use in the diagnosis, prophylaxis, treatmentand/or symptomatic relief of Picornaviridae infections in vertebrateanimals.

Preferably, the Picornaviridae virus of the present invention is fromthe genus Rhinovirus or Enterovirus.

Accordingly, the present invention contemplates a method forameliorating the effects of Rhinovirus or Enterovirus infection in avertebrate animal, said method comprising administering to said animalan effective amount of one or more compounds selected from the compoundsof Formulae I-TV or a parent compound thereof.

Amiloride is an example of a parent compound of EIPA.

More particularly, the present invention contemplates a method forameliorating the effects of Rhinovirus or Enterovirus infection in avertebrate animal, said method comprising administering to said animalan effective amount of one or more compounds selected from the compoundsof Formulae V-IX.

Even more particularly, the viral species from the genera Rhinovirus andEnterovirus are Echovirus 11 (EV11), Coxsackievirus B3 (CVB3) andRhinovirus 2 (RV2) and Rhinovirus 14 (RV14).

As stated above, a “vertebrate animal” includes a primate, human,livestock animal (e.g. sheep, horse, cow, donkey, pig, goat), laboratorytest animal (e.g. mouse, rabbit, guinea pig), companion animal (e.g.cat, dog) as well as avian, reptilian and amphibian species. The mostpreferred vertebrate animal is a human. The vertebrate animal of thepresent invention may be referred to herein as a subject.

Accordingly, the present invention contemplates a method forameliorating the effects of Picomaviridae infection in a human subject,said method comprising administering to said human subject an effectiveamount of one or more compounds selected from the compounds of FormulaeI-IV or a parent compound thereof.

Accordingly, the present invention contemplates a method forameliorating the effects of Picomaviridae infection in a human subject,said method comprising administering to said human subject an effectiveamount of one or more compounds selected from the compounds of FormulaeV-IX.

Accordingly, the present invention contemplates a method forameliorating the effects of Rhinovirus or Enterovirus infection in ahuman subject, said method comprising administering to said humansubject an effective amount of one or more compounds selected from thecompounds of Formulae I-IV or a parent compound thereof.

Accordingly, the present invention contemplates a method forameliorating the effects of Rhinovirus or Enterovirus infection in ahuman subject, said method comprising administering to said humansubject an effective amount of one or more compounds selected from thecompounds of Formulae I-IX or a parent compound thereof.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound of any of theFormulae (I-IV) is modified by making acid or base salts of the compoundof Formulae (I-IV), respectively. Examples of pharmaceuticallyacceptable salts include, but are, not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts oracidic residues such as carboxylic acids; and the like.

The compounds of the present invention may also be in the form ofprodrugs.

“Prodrugs” are considered to be any covalently bonded carriers whichrelease the active parent drug according to any of Formulae I-IV or aparent compound thereof or V-EX in vivo when such prodrug isadministered to a vertebrate animal subject. Prodrugs of the compoundsof Formulae I-IV or V-IX or a parent compound thereof are prepared bymodifying functional groups present in the compounds in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compounds. Prodrugs include compounds of FormulaeI-IV wherein hydroxy, amine, or sulfhydryl groups are bonded to anygroup that, when administered to a subject, cleaves to form a freehydroxyl, amino or sulfhydryl group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate, or benzoatederivatives of alcohol and amine functional groups in the compounds ofFormulae I-IV; phosphate esters, dimethylglycine esters and carboxyalkylesters of alcohol and phenol functional groups in the compounds ofFormulae I-IV; and the like.

The pharmaceutically acceptable salts of the compounds of Formulae I-IVor a parent compound thereof include the conventional non-toxic salts orthe quaternary ammonium salts of the compounds of Formulae I-IV or aparent compound thereof formed, for example, from non-toxic inorganic ororganic acids. For example, such conventional non-toxic salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the compounds of Formulae I-IV or a parent compoundthereof which contain a basic or acidic moiety by conventional chemicalmethods. Generally, such salts can be prepared by reacting the free acidor base forms of these compounds with a stoichiometric amount of theappropriate base or acid ill water or in an organic solvent, or in amixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)ed., Mack Publishing Company, Easton, Pa., 1985, p 1418, the disclosureof which is hereby incorporated by reference.

The term “treatment” is used in its broadest sense and includes theprevention of a disease condition as well as facilitating theamelioration of the effects of the signs and symptoms of a disease.

The term “prophylaxis” is also used herein in its broadest sense toencompass a reduction in the risk of development of a disease. Incertain conditions, an agent may act to treat a subjectprophylactically. Furthermore, the prophylactic administration of anagent may result in the agent becoming involved in the treatment of thedisease condition. Use of the terms “treatment” or “prophylaxis” is notto be taken as limiting the intended result which is to reduce theadverse effects of a disease or to potentiate the immune system'sresponse or components therein to ameliorate the signs and/or symptomsor risk of development of the signs and/or symptoms cause or facilitatedby a disease.

The present invention further extends to pharmaceutical compositionsuseful in the treatment of a disease condition comprising the chemicalcompound or compounds of the present invention. In this regard, thechemical agents of the invention can be used as actives for thetreatment or prophylaxis of a disease condition such as rhinovirus. Thechemical agents can be administered to a subject either by themselves,or in pharmaceutical compositions where they are mixed with a suitablepharmaceutically acceptable carrier.

An effective amount is administered. An effective amount includes atherapeutically effective amount which is an amount effective toinhibit, reduce or otherwise retard viral replication, processing and/orattachment or release. In one embodiment, the therapeutically effectiveamount is an amount which inhibits, blocks or at least partially impairsan ion channel. The effective amount, therefore, may be an ion channelblocking effective amount. The ability for a compound to block an ionchannel may be readily observed by the downstream effect on viralreplication and/or processing.

Accordingly, the invention also provides a composition for treatmentand/or prophylaxis of viral infection comprising one or more chemicalcompounds as defined herein by Formulae I-IV or a parent compoundthereof, together with one or more pharmaceutically acceptable carriersand/or diluents.

Depending on the specific conditions being treated, chemical agents maybe formulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, (supra). Suitable routes may, for example,include oral, rectal, transmucosal, or intestinal administration; nasalspray, aerosol delivery, parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. For injection, the chemical agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart. Intra-muscular and subcutaneous injection is appropriate, forexample, for administration of immunomodulatory compositions andvaccines.

The chemical agents can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the invention tobe formulated in dosage forms such as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated. These carriers may be selected from sugars,starches, cellulose and its derivatives, malt, gelatine, talc, calciumsulphate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline, andpyrogen-free water.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve their intended purpose. The dose of agentadministered to a patient should be sufficient to effect a beneficialresponse in the patient over time such as a reduction in the symptomsassociated with the presence of an inflammatory condition in a subject.The quantity of the agent(s) to be administered may depend on thesubject to be treated inclusive of the age, sex, weight and generalhealth condition thereof. In this regard, precise amounts of theagent(s) for administration will depend on the judgement of thepractitioner. In determining the effective amount of the chemical agentto be administered in the treatment or prophylaxis of a diseasecondition, the physician may evaluate progression of the disorder. Inany event, those of skill in the art may readily determine suitabledosages of the chemical agents of the invention.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Such compositions may beprepared by any of the methods of pharmacy but all methods include thestep of bringing into association one or more chemical agents asdescribed above with the carrier which constitutes one or more necessaryingredients. In general, the pharmaceutical compositions of the presentinvention may be manufactured in a manner that is itself known, e.g. bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilisingprocesses.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

Dosage forms of the chemical agents of the invention may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of an agent of theinvention may be effected by coating the same, for example, withhydrophobic polymers including acrylic resins, waxes, higher aliphaticalcohols, polylactic and polyglycolic acids and certain cellulosederivatives such as hydroxypropylmethyl cellulose. In addition,controlled release may be effected by using other polymer matrices,liposomes and/or microspheres.

For any chemical agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays such as to reduce or ameliorate the symptoms of infectionin vitro or to potentiate immune cells in vitro. For example, a dose canbe formulated in animal models to achieve a circulating concentrationrange that includes the IC50 as determined in cell culture (e.g. theconcentration of a test agent, which achieves a half-maximal inhibitionof infection). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of such chemical agents can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g. for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies are used in formulating a range of dosages for use in humans.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (see for exampleFingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 p1,1975).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active agent which are sufficient to maintainsymptom-ameliorating effects. Usual patient dosages for systemicadministration range from 1-2000 mg/day, commonly from 1-250 mg/day, andtypically from 10-150 mg/day. Stated in terms of patient body weight,usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patientbody surface areas, usual dosages range from 0.5-1200 mg/m²/day,commonly from 0.5-150 mg/m²/day, typically from 5-100 mg/m²/day.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a tissue, often in a depot or sustained release formulation.Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thetissue. In cases of local administration or selective uptake, theeffective local concentration of the agent may not be related to plasmaconcentration.

The chemical agents of the invention can also be delivered topically.For topical administration, a composition containing between 0.001-5% ormore chemical agent is generally suitable. Regions for topicaladministration include the skin surface and also mucous membrane tissuesof the vagina, rectum, nose, mouth, and throat. Compositions for topicaladministration via the skin and mucous membranes should not give rise tosigns of irritation, such as swelling or redness.

The topical composition may include a pharmaceutically acceptablecarrier adapted for topical administration. Thus, the composition maytake the form of a suspension, solution, ointment, lotion, sexuallubricant, cream, foam, aerosol, spray, suppository, implant, inhalant,tablet, capsule, dry powder, syrup, balm or lozenge, for example.Methods for preparing such compositions are well known in thepharmaceutical industry.

In one embodiment, the topical composition is administered topically toa subject, e.g. by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch”. Such compositions include, for example, lotions, creams,solutions, gels and solids. Suitable carriers for topical administrationpreferably remain in place on the skin as a continuous film, and resistbeing removed by perspiration or immersion in water. Generally, thecarrier is organic in nature and capable of having dispersed ordissolved therein a chemical agent of the invention. The carrier mayinclude pharmaceutically-acceptable emollients, emulsifiers, thickeningagents, solvents and the like.

The present invention is described with reference to the followingnon-limiting Example.

EXAMPLE

Representative viruses of the Rhinovirus and Enterovirus genera werechosen for study: Rhinovirus 2 (RV2), Rhinovirus 14 (RV14),Coxsackievirus BE (CVB3) and Echovirus 11 (EV11). Each virus ispropogated in two different cell lines as follows: RV2 and RV14 in HELand HeLa cells, CVB3 in BS-C-1 and HeLa (human cervical adenocarcinoma)cells, EV11 in BS-C-1 (African green monkey kidney, primary) and HEL(human embryonic lung, primary) cells. Propogation of virus strains intwo different cell lines is performed as different cell types mayutilize different ion transport pathways.

Cells are infected with low levels of virus (0.01 plaque formingunit/cell) to obtain multiple infection cycles in the course of anexperiment. Multiple infection cycles allow the detection of antiviralactivity of a compound regardless of what step of the infection cycle itaffects. Cells inoculated with the viruses are incubated in culturemedia containing different concentrations of the test compounds anddose-response curves are obtained for all compound/virus/cell typecombinations. Virus yields are measured by plaque assay at the end ofthe experiment, and the reduction of virus yield in compound-treatedsamples compared with untreated cells is calculated. Cytotoxicity of thecompounds is measured in parallel experiments of uninfected cells usingthe metabolic dye Alamar Blue as an indicator.

Antiviral assays are determined as follows. Monolayers of HeLa T cells(human cervical adenocarcinoma) in 12-well plates are infected with 0.01plaque forming units per cell of Rhinovirus 2 in minimum essentialmedium with Earle's salts (MEM) supplemented with 1% v/v fetal bovineserum (FBS) or mock-infected with medium for 1 hour. The inoculum isthen replaced with fresh medium containing the ion transport blockers(Verapamil, 5-(N-ethyl-N-isopropyl)amiloride [EIPA], Econazole, Benzamilor Amiloride) in concentrations ranging from 550 μM to 0 μM (no drugcontrol) in 2-fold dilutions. Cells are further incubated for 70 hoursat 34° C., until extensive cell death is observed in the no drugcontrol. Cells plus culture supernatants are freeze-thawed and virustitre in each sample is determined by plaque assay.

As a control, the cytotoxicity of ion transport blockers is tested oncells.

Toxicity of the compounds on HeLa T cells is evaluated in an experimentwhich runs parallel to the effect on viral production, using uninfectedcells. Cells are incubated in MEM (1% v/v FBS) containing the iontransport blockers: Verapamil, 5-(N-ethyl-N-isopropyl)amiloride [EIPA],Econazole, Benzamil or Amiloride in concentrations ranging from 550 μMto 0 μM for 70 hours at 34° C. inside 12-well plates. The cells are thenrinsed with 10 mM Tris-HCl, 150 mM NaCl, pH 7.5, and incubated for 1hour at 34° C. in 500 μl/well of 10% solution of colourimetric indicatorof metabolic activity Alamar Blue (Serotec) in MEM (1% v/v FBS).Following this step, the absorbance (A570-A600) of culture supernatantswas read on a spectrophotometer (Pharmacia Biotech). Under theseconditions the absorbance values are proportional to the metabolicactivity of cells in each sample. The effects of EIPA and Verapamil onHeLa cells are shown in FIGS. 1 and 2 and FIGS. 3 and 4, respectively.Compared to the inhibition by these compounds of virus production, thecompounds were far less toxic to HeLa cells. The effects of Amilorideand Benzamil on Coxsackievirus B3 (an Enterovirus) in HeLa cells areshown in FIGS. 5 and 6.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of anly two or more of said steps or features.

BIBLIOGRAPHY

-   Monto et al., Clin. Ther. 10: 1615-27, 2001;-   Turner, Antiviral Res. 49(1): 1-14, 2001;-   Irurzum et al., J Virol. 69(8): 5142-6, 1995;-   van Kuppeveld et al., EMBO J 16(12): 3519-32,1997;

1. A method for controlling spread of Picomaviridae infection in avertebrate animal said method comprising administering to said animal aneffective amount of one or more compounds selected from the compounds ofFormulae I-IV or a parent of these compounds:

where n is 0-10 atoms and both n and X may be the same or different andeach is selected from carbon, oxygen, nitrogen, sulfur, phosphorus,silicon, boron, arsenic and selenium; R₁ to R₂₃ may be the same ordifferent and each is selected from hydrogen, F, Cl, Br, I, CN, NC, NO₂,CF₃, COR₁, CO₂R₁, OR₁, SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁,SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂,(C═X)R₁ or X(C═X)R₁ where X is selected from sulfur, oxygen andnitrogen; C₁-C₂₀ alkyl (branched and/or straight chained), C₁-C₂₀arylalkyl, C₃-C₈ cycloalkyl, C₁-C₁₀ aldoxy, C₁-C₁₀ alkyl carbonyl,C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle, C₂-C₁₀ alkenyl,C₁-C₁₀ heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ [CN, NC, OR₁, SR₁, NR₁R₂,N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂,P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂]alkyl; aryl is C₆-C₁₄ with any modeof substitution containing F, Cl, Br, I, NO₂, CF₃, CN, NC, COR₁, CO₂R₁,OR₁, SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂,SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂]alkyl.Heteroaryl is oxazolyl, thiazaoyl, thienyl, furyl, 1-isobenzofuranyl,3H-pyrrolyl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl,isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl,indolizinyl, isoindolyl, indoyl, indolyl, purinyl, phthalazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl,azepinyl, oxepinyl, thiepinyl, benzofuranyl, isobenzofuranyl,thionaphthenyl, isothionaphthenyl, indoleninyl, 2-isobenzazolyl,1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, isoindazolyl, indoxazinyl,benzoxazolyl, anthranilyl, quinolinyl, isoquinolinyl, cinnolinyl,quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl,pyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl.
 2. The method of claim 1wherein the compounds are administered for a time and under conditionssufficient to reduce the amount of virus replication and/or release fromcells exposed to said compounds.
 3. The method of claim 1 or 2 whereinthe compounds are administered with a pharmaceutically acceptablecounterion.
 4. The method of claim 3 wherein the counterion is selectedfrom hydrochloric acid, sulphuric acid, acetic acid, lactic acid,tartaric acid, malic acid and succinic acid.
 5. The method of claim 1wherein the compounds block an ion transport pathway.
 6. The method ofclaim 1 wherein the compound is Verapamil or a functional derivativethereof.
 7. The method of claim 1 wherein the compound is Econazole or afunctional derivative thereof.
 8. The method of claim 1 wherein thecompound is Benzamil or a functional derivative thereof.
 9. The methodof claim 1 wherein the compound is 5-(N-ethyl-N-iospropyl) amiloride(EIPA) or a functional derivative thereof.
 10. The method of claim 1wherein the compound is Amiloride or a functional derivative thereof.11. The method of claim 1 or 6 or 7 or 8 or 9 or 10 wherein thevertebrate animal is a mammal.
 12. The method of claim 11 wherein themammal is a human.
 13. The method of claim 1 wherein the Picornaviridaevirus is Rhinovirus.
 14. The method of claim 1 wherein thePicornaviridae virus is Enterovirus.
 15. The method of claim 13 whereinthe Rhinovirus is selected from bovine rhinovirus 1, 2 and 3, humanrhinovirus 1A and human rhinovirus 1 to
 100. 16. The method of claim 14wherein the Enterovirus is selected from bovine enterovirus 1 and 2,human Coxsackievirus A1 to 22, human Coxsackievirus A24, humanCoxsackievirus B1 to 6, human echovirus 1 to 7, 9, 11 to 27 and 29 to33, human enterovirus 68 to 71, human poliovirus 1, 2 and 3, porcineenterovirus, simian enterovirus 1 to 18 and Vilyuisk virus.
 17. Use of acompound which blocks an ion transport pathway which pathway results inan influx of ions into a cell following infection with a Picornaviridaevirus in the manufacture of a medicament for the treatment of viralinfection by a Picomaviridae virus in a vertebrate animal.
 18. Use ofclaim 17 wherein the vertebrate animal is a mammal.
 19. Use of claim 18wherein the mammal is a human.
 20. Use of claim 17 or 18 or 19 whereinthe compound is selected from a compound of Formula I-IV or a parent ofthese compounds:

where n is 0-10 atoms and both n and X may be the same or different andeach is selected from carbon, oxygen, nitrogen, sulfur, phosphorus,silicon, boron, arsenic and selenium; R₁ to R₂₃ may be the same ordifferent and each is selected from hydrogen, F, Cl, Br, I, CN, NC, NO₂,CF₃, COR₁, CO₂R₁, OR₁, SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁,SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂,(C═X)R₁ or X(C═X)R₁ where X is selected from sulfur, oxygen andnitrogen; C₁-C₂₀ alkyl (branched and/or straight chained), C₁-C₂₀arylalkyl, C₃-C₈ cycloalkyl, C₁-C₁₀ aldoxy, C₁-C₁₀ alkyl carbonyl,C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, C₁-C₁₄ heterocycle, C₂-C₁₀ alkenyl,C₁-C₁₀ heteroarylalkyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ haloalkyl,dihaloalkyl, trihaloalkyl, haloalkoxy, C₁-C₁₀ [CN, NC, OR₁, SR₁, NR₁R₂,N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂, SO₂NR₁R₂, SO₃NR₁R₂,P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂]alkyl; aryl is C₆-C₁₄ with any modeof substitution containing F, Cl, Br, 1, NO₂, CF₃, CN, NC, COR₁, CO₂R₁,OR₁, SR₁, NR₁R₂, N(═O)₂, NR₁OR₂, ONR₁R₂, SOR₁, SO₂R₁, SO₃R₁, SONR₁R₂,SO₂NR₁R₂, SO₃NR₁R₂, P(R₁)₃, P(═O)(R₁)₃, Si(R₁)₃, B(R₁)₂]alkyl.Heteroaryl is oxazolyl, thiazaoyl, thienyl, furyl, 1-isobenzofuranyl,3H-pyrrolyl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl,isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl,indolizinyl, isoindolyl, indoyl, indolyl, purinyl, phthalazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl,azepinyl, oxepinyl, thiepinyl, benzofuranyl, isobenzofuranyl,thionaphthenyl, isothionaphthenyl, indoleninyl, 2-isobenzazolyl,1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, isoindazolyl, indoxazinyl,benzoxazolyl, anthranilyl, quinolinyl, isoquinolinyl, cinnolinyl,quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl,pyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl.
 21. Use of claim 20wherein the compound is selected from Verapamil, Benzamil, Econazol,EIPA and Amiloride or a derivative thereof or a pharmaceuticallyacceptable salt thereof.
 22. Use of claim 21 wherein the Picomaviridaevirus is Rhinovirus or Enterovirus.
 23. A method for controlling spreadof Rhinovirus or Enterovirus infection in a vertebrate animal, saidmethod comprising administering to said animal an effective amount ofone or more compounds selected from the compounds of Formulae I-IV or aparent compound thereof.
 24. The method of claim 23 wherein the compoundis selected from Verapamil, Benzamil, Econazol, EIPA and Amiloride. 25.The method of claim 23 or 24 wherein the vertebrate animal is a mammal.26. The method of claim 25 wherein the mammal is a human.
 27. Apharmaceutical preparation when used to control spread of Picomaviridaeinfection according to the method of claim 1 comprising one or morecompounds selected from Verapamil, Benzamil, Econazol, EIPA andAmiloride or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier and/or diluent.